Curli Fibers Research Articles

Epigallocatechin gallate protects mice from Salmonella enterica ser. Typhimurium infection by modulating bacterial virulence through quorum sensing inhibition.

Salmonella enterica ser. Typhimurium is a common pathogen that poses a considerable public health threat, contributing to severe gastrointestinal diseases and widespread foodborne illnesses. The virulence of S. Typhimurium is regulated by quorum sensing (QS) and the type III secretion system (T3SS). This study investigated the inhibitory effects and anti-QS activity of epigallocatechin gallate (EGCG), which is a bioactive ingredient found in green tea, on the virulence of S. Typhimurium. In vitro bacterial experiments demonstrated that EGCG inhibited the production of autoinducers, biofilm formation, and flagellar activity by downregulating the expression of AI-1, AI-2, Salmonella pathogenicity islands (SPI)-1, SPI-2, and genes related to flagella, fimbriae, and curli fibers. In a mouse model of S. Typhimurium-induced enteritis, EGCG considerably reduced intestinal colonization by S. Typhimurium and alleviated intestinal damage. In conclusion, EGCG protects the intestines of mice infected with S. Typhimurium by inhibiting QS-induced virulence gene expression, demonstrating its potential as a therapeutic agent for controlling S. Typhimurium infections.

Contact detection between curved fibres: high order makes a difference

Computer Graphics has a long history in the design of effective algorithms for handling contact and friction between solid objects. For the sake of simplicity and versatility, most methods rely on low-order primitives such as line segments or triangles, both for the detection and the response stages. In this paper we carefully analyse, in the case of fibre systems, the impact of such choices on the retrieved contact forces. We highlight the presence of artifacts in the force response that are tightly related to the low-order geometry used for contact detection. Our analysis draws upon thorough comparisons between the high-order super-helix model and the low-order discrete elastic rod model. These reveal that when coupled to a low-order, segment-based detection scheme, both models yield spurious jumps in the contact force profile. Moreover, these artifacts are shown to be all the more visible as the geometry of fibres at contact is curved. In order to remove such artifacts we develop an accurate high-order detection scheme between two smooth curves, which relies on an efficient adaptive pruning strategy. We use this algorithm to detect contact between super-helices at high precision, allowing us to recover, in the range of wavy to highly curly fibres, much smoother force profiles during sliding motion than with a classical segment-based strategy. Furthermore, we show that our approach offers better scaling properties in terms of efficiency vs. precision compared to segment-based approaches, making it attractive for applications where accurate and reliable forces are desired. Finally, we demonstrate the robustness and accuracy of our fully high-order approach on a challenging hair combing scenario.

Genetically Decorating Microbial Surface Curli Fiber with Biocatalytic Properties

Genetically Decorating Microbial Surface Curli Fiber with Biocatalytic Properties

Weak Hydrogen Bonds in Temporary Shape Changes of Curly Human Hair Fibers: Preliminary Evidence

Weak Hydrogen Bonds in Temporary Shape Changes of Curly Human Hair Fibers: Preliminary Evidence

Synthetic silica fibers of different length, diameter and shape: synthesis and interaction with rat (NR8383) and human (THP-1) macrophages in vitro, including chemotaxis and gene expression profile

BackgroundInhalation of biopersistent fibers like asbestos can cause strong chronic inflammatory effects, often resulting in fibrosis or even cancer. The interplay between fiber shape, fiber size and the resulting biological effects is still poorly understood due to the lack of reference materials.ResultsWe investigated how length, diameter, aspect ratio, and shape of synthetic silica fibers influence inflammatory effects at doses up to 250 µg cm-2. Silica nanofibers were prepared with different diameter and shape. Straight (length ca. 6 to 8 µm, thickness ca. 0.25 to 0.35 µm, aspect ratio ca. 17:1 to 32:1) and curly fibers (length ca. 9 µm, thickness ca. 0.13 µm, radius of curvature ca. 0.5 µm, aspect ratio ca. 70:1) were dispersed in water with no apparent change in the fiber shape during up to 28 days. Upon immersion in aqueous saline (DPBS), the fibers released about 5 wt% silica after 7 days irrespectively of their shape. The uptake of the fibers by macrophages (human THP-1 and rat NR8383) was studied by scanning electron microscopy and confocal laser scanning microscopy. Some fibers were completely taken up whereas others were only partially internalized, leading to visual damage of the cell wall. The biological effects were assessed by determining cell toxicity, particle-induced chemotaxis, and the induction of gene expression of inflammatory mediators.ConclusionsStraight fibers were only slightly cytotoxic and caused weak cell migration, regardless of their thickness, while the curly fibers were more toxic and caused significantly stronger chemotaxis. Curly fibers also had the strongest effect on the expression of cytokines and chemokines. This may be due to the different aspect ratio or its twisted shape.

Development and characterization of a bacterial enzyme cascade reaction system for efficient and stable PET degradation

The widespread use of polyethylene terephthalate (PET) in various industries has led to a surge in microplastics (MPs) pollution, posing a significant threat to ecosystems and human health. To address this, we have developed a bacterial enzyme cascade reaction system (BECRS) that focuses on the efficient degradation of PET. This system harnesses the Escherichia coli (E. coli) surface to display CsgA protein, which forms curli fibers, along with the carbohydrate-binding module 3 (CBM3) and PETases, to enhance the adsorption and degradation of PET. The study demonstrated that the BECRS achieved a notable PET film degradation rate of 3437 ± 148 μg/(d*cm²), with a degradation efficiency of 21.40% for crystalline PET MPs, and the degradation products were all converted to TPA. The stability of the system was evidenced by retaining over 80% of its original activity after multiple uses and during one month of storage. Molecular dynamics simulations confirmed that the presence of CsgA did not interfere with the enzymatic activity of PETases. This BECRS represents a significant step forward in the biodegradation of PET, particularly microplastics, offering a practical and sustainable solution for environmental pollution control.

A novel F type plasmid encoding mcr-10 in a clinical Enterobacter ludwigii strain from a tertiary hospital in the Czech Republic

ObjectiveHere we describe a novel IncFIA plasmid harboring mcr-10 gene in a clinical Enterobacter ludwigii strain isolated at the University Hospital in Pilsen in the Czech Republic. MethodsThe strain was subjected to antibiotic susceptibility testing. Whole genome sequencing was performed using Illumina for short read sequencing and Oxford Nanopore Technologies for long-read sequencing followed by hybrid assembly. The resulting genome was used to detect species using average nucleotide identity, resistance genes, plasmid replicon and MLST (using center for genomic epidemiology databases; ResFinder, PlasmidFinder and MLST repsectively) and virulence genes using VFDB. ResultsΤhe strain showed susceptibility against tetracycline, cefuroxime and chloramphenicol, and was susceptible to the 2nd and 3rd generation of cephalosporins, carbapenems and colistin. Genome analysis identified the strain as E. ludwigii sequence type ST20 and located the mcr-10 gene on an IncFIA (HI1)/IncFII (Yp) plasmid (pI9455333_MCR10; 129,863 bp). Upon blasting the nucleotide sequence of pI9455333_MCR10 against NCBI database; no similar plasmid sequence was detected implying a novel plasmid structure. Nevertheless, it showed a partial similarity with: pRHBSTW-00123_3 and FDAARGOS 1432 which were detected in Enterobacter cloacae complex (ECC) strains in wastewater samples in 2017 in UK and in 2021 in the USA respectively, and pEC81-mcr which was detected in a clinical Escherichia coli strain in 2020 in China. Moreover, I9455333cz genome carried virulence genes coding for: curli fibers, fimbrial adherence determinants, siderophore aerobactin, iron uptake proteins, and regulators of sigma factor. ConclusionIn conclusion, we identified a novel IncF plasmid harboring mcr-10 gene in a clinical Enterobacter ludwigii strain. To our knowledge, this is the first clinical report of mcr-10 in the Czech Republic.

Electrospun 3D Curly Electret Nanofiber Air Filters for Particulate Pollutants

Amidst rapid industrialization and urbanization, air pollution has emerged as a global environmental challenge. Traditional air filtration materials face challenges in effectively filtering PM0.3 and often result in discomfort due to high air resistance when used for personal protection, as well as excessive energy consumption in industrial air purification applications. This study initially utilized extremely high environmental humidity to induce fiber formation, resulting in the preparation of a fluffy fiber membrane with a three-dimensional curly morphology, which increased the porosity to 96.93%, significantly reducing air resistance during filtration. Subsequently, rutile TiO2 with a high dielectric constant was introduced, exploiting the low pressure drop characteristic of the fluffy 3D curly fiber membrane combined with the electret effect of TiO2 nanoparticles to notably improve the issue of excessive pressure drops while maintaining filtration efficiency. The microstructure, morphology, and element distribution of the fibers were analyzed using FESEM and EDS. FTIR and XRD were employed to examine the functional groups and crystal structure within the fibers. The electret effect and filtration performance of the fiber membrane were investigated using an electrostatic tester and a particulate filtration efficiency tester. The results demonstrated that inducing fiber formation under high-humidity conditions could produce fibers with a 3D curly structure. The fiber membrane was highly fluffy, significantly reducing the pressure drop. Introducing an appropriate amount of titanium dioxide markedly improved the electrostatic effect of the fiber membrane, enhancing the filtration performance of the 3D curly PVDF/TiO2 composite fiber membrane. With a 0.5% addition of TiO2 nanoparticles, the filtration efficiency of the fiber membrane reached approximately 99.197%, with a pressure drop of about 49.83 Pa. This study offers a new approach to developing efficient, low-resistance air filtration materials, showcasing the potential of material innovation in addressing air quality challenges within the sustainable development framework.

Endowing textiles with self-repairing ability through the fabrication of composites with a bacterial biofilm

To address the increasing environmental footprint of the fast-growing textile industry, self-repairing textile composites have been developed to allow torn or damaged textiles to restore their morphological, mechanical, and functional features. A sustainable way to create these textile composites is to introduce a coating material that is biologically derived, biodegradable, and can be produced through scalable processes. Here, we fabricated self-repairing textile composites by integrating the biofilms of Escherichia coli (E. coli) bacteria into conventional knitted textiles. The major structural protein component in E. coli biofilm is a matrix of curli fibers, which has demonstrated extraordinary abilities to self-assemble into mechanically strong macroscopic structures and self-heal upon contact with water. We demonstrated the integration of biofilm through three simple, fast, and scalable methods: adsorption, doctor blading, and vacuum filtration. We confirmed that the composites were breathable and mechanically strong after the integration, with improved Young’s moduli or elongation at break depending on the fabrication method used. Through patching and welding, we showed that after rehydration, the composites made with all three methods effectively healed centimeter-scale defects. Upon observing that the biofilm strongly attached to the textiles by covering the extruding textile fibers from the self-repair failures, we proposed that the strength of the self-repairs relied on both the biofilm’s cohesion and the biofilm-textile adhesion. Considering that curli fibers are genetically-tunable, the fabrication of self-repairing curli-expressing biofilm-textile composites opens new venues for industrially manufacturing affordable, durable, and sustainable functional textiles.

Mapping direct and indirect MarA/SoxS/Rob/RamA regulons in Salmonella Typhimurium reveals repression of csgD and biofilm formation.

The closely related transcription factors MarA, SoxS, Rob and RamA control overlapping stress responses in many enteric bacteria. Furthermore, constitutive expression of such regulators is linked to clinical antibiotic resistance. In this work we have mapped the binding of MarA, SoxS, Rob and RamA across the Salmonella Typhimurium genome. In parallel, we have monitored changes in transcription start site use resulting from expression of the regulators. Together, these data allow direct and indirect gene regulatory effects to be disentangled. Promoter architecture across the regulon can also be deduced. At a phylogenetic scale, around one third of regulatory targets are conserved in most organisms encoding MarA, SoxS, Rob or RamA. We focused our attention on the control of csgD, which encodes a transcriptional activator responsible for stimulating production of curli fibres during biofilm formation. We show that expression of csgD is particularly sensitive to SoxS that binds upstream to repress transcription. This differs to the situation in Escherichia coli, where MarA regulates csgD indirectly.

Highly efficiency production of D-allulose from inulin using curli fiber multi-enzyme cascade catalysis

D-Allulose is a rare sugar with numerous physiological benefits and low caloric content, which can be obtained from inulin through enzymatic catalysis. In this study, we combined D-allulose 3-epimerase, exo- and endo-inulinases (EXINU and ENINU) with the NGTag/NGCatcher/CsgA system to accelerate D-allulose accumulation from inulin. Molecular dynamics simulations were used to screen linkers of appropriate length for ENINU. In vitro, we successfully observed the assembled NGCatcher_ENINU_CsgA, NGTag_EXINU, and DAERK fibers using fluorescent labelling with GFP, YFP, and mCherry. The optimal pH and temperature of the tagged variants were comparable to those of the wild-type, and the MD simulations showed that NGCatcher_ENINU_CsgA had improved stability in the working environment of EXINU. D-Allulose accumulation rate of the assembled enzymes cascade (NGCatcher_ENINU_CsgA/NGTag_EXINU_CsgA/DAERK) reached 0.25 g/L min−1 (1.25 mgD-allulose mgDAERK−1 min−1) at an inulin concentration of 100 g/L. The assembled system greatly improves the high-valued productions of rare sugars from cheap biomass.

Salmonella enterica serovar Typhimurium STM1266 encodes a regulator of curli biofilm formation: the brfS gene.

The major biofilm pathway in Salmonella enterica serovar Typhimurium involves specific growth conditions that induce the csgA gene whose product forms surface curli fibers that mediate biofilm formation. We have found that the previously uncharacterized STM1266 gene in S. Typhimurium plays a role in regulating biofilm formation via the curli pathway. S. Typhimurium ΔSTM1266 strains display a biofilm defect, and overexpression of STM1266 results in enhanced biofilm formation. STM1266 deletion resulted in lowered csgA expression using promoter-reporter β-galactosidase assays, and csgA and csgD deletions abrogate the effects of STM1266 overexpression on biofilm formation while deletion of bcsA (encoding an essential enzyme for cellulose formation) has no effect. In a mouse infection model, the ΔSTM1266 strain displayed results similar to those seen for previously reported ΔcsgA strains. The STM1266 gene is predicted to encode a DNA-binding transcriptional regulator of the MerR family and is homologous to the Escherichia coli BluR regulator protein. We respectfully propose to ascribe the name brfS (biofilm regulator for Salmonella Typhimurium) to the STM1266 gene.

Bionic artificial penile Tunica albuginea

Bionic artificial penile Tunica albuginea

Anti-biofilm activity of caffeine against uropathogenic E. coli is mediated by curli biogenesis

Biofilms are assemblages of sessile microorganisms that form an extracellular matrix around themselves and mediate attachment to surfaces. The major component of the extracellular matrix of Uropathogenic E. coli and other Enterobacteriaceae are curli fibers, making biofilms robust and resistant to antimicrobials. It is therefore imperative to screen antibiofilm compounds that can impair biofilm formation. In the present study, we investigated the curli-dependent antibiofilm activity of caffeine against UPEC strain CFT073 and commensal strain E. coli K-12MG1655.Caffeine significantly reduced the biofilm formation of both UPEC and E. coli K-12 by 86.58% and 91.80% respectively at 48 mM caffeine as determined by Crystal Violet assay. These results were further confirmed by fluorescence microscopy and Scanning Electron Microscope (SEM). Caffeine significantly reduced the cytotoxicity and survivability of UPEC. Molecular docking analysis revealed a strong interaction between caffeine and curli regulator protein (Csg D) of E. coli. The qRT-PCR data also showed significant downregulation in the expression of CsgBA and the CsgDEFG operon at both 24 mM and 48 mM caffeine. The findings revealed that caffeine could inhibit E. coli biofilm formation by regulating curli assembly and thus may be used as an alternative therapeutic strategy for the treatment of chronic E. coli biofilm-related infections.

Analytical interpretation of microscale fiber deviation in designing for polymer melt electrohydrodynamic-based additive manufacturing

Aided by translational material collection, melt electrowriting is an additive electrohydrodynamic-based materials processing technique capable of fabricating fibrous 3D structured materials with customizable microscale architectures suitable for spatially defined tissue models. An inherent phenomenon of the melt electrowriting process is the characteristic jet lag, which results in spatial differences between the nozzle position ( np ) and the jet contact point ( cp ). Moreover, cp does not deterministically follow the np trajectory when the movement of np relative to the collector is directionally altered. Instead, fiber deviation related to the toolpath deteriorates the printing accuracy and is ubiquitous for curly fiber printing. In order to address this bottleneck, an analytical construct informed by fiber placement prediction and control is advanced. Specifically, by way of vector analysis and differential geometry, the position and speed relationships between np and cp are found to be governed by position- and speed-matching equations. Representations of these equations can be elaborated into the specific cases of straight and steady curly fiber printing, which are investigated and verified herein. Finally, the real-time identification of cp as well as dynamic control of translational stage speed are identified as critical steps towards reliable implementation of the toolpath design in curly fiber printing.

Single domain antibodies against enteric pathogen virulence factors are active as curli fiber fusions on probiotic E. coli Nissle 1917.

Enteric microbial pathogens, including Escherichia coli, Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic E. coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E. coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro. The probiotic strain E. coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E. coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen (Shigella flexneri), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eukaryotic pathogen (Cryptosporidium parvum) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics.

Editors' Note: Association of Fecal and Plasma Levels of Short-Chain Fatty Acids With Gut Microbiota and Clinical Severity in Patients With Parkinson Disease

In “Association of Fecal and Plasma Levels of Short-Chain Fatty Acids With Gut Microbiota and Clinical Severity in Patients With Parkinson Disease,” Chen et al. reported that a reduction in fecal short-chain fatty acids (SCFAs) is associated with increased clinical severity of Parkinson disease (PD). Brenner commented that this may be attributed to the fact that a reduction in SCFAs can lead to increased growth of Escherichia coli , which produce curli fibers that can accelerate production of amyloids and subsequent action pathology related to alpha-synuclein. Lin et al. agreed that curli production may lead to alpha-synuclein aggregation and neuroinflammation, as seen in PD. The authors further concurred that the gut-brain relationship in PD may be related to SCFAs, curli-producing E. coli , functional amyloids, and alpha-synuclein. In “Association of Fecal and Plasma Levels of Short-Chain Fatty Acids With Gut Microbiota and Clinical Severity in Patients With Parkinson Disease,” Chen et al. reported that a reduction in fecal short-chain fatty acids (SCFAs) is associated with increased clinical severity of Parkinson disease (PD). Brenner commented that this may be attributed to the fact that a reduction in SCFAs can lead to increased growth of Escherichia coli , which produce curli fibers that can accelerate production of amyloids and subsequent action pathology related to alpha-synuclein. Lin et al. agreed that curli production may lead to alpha-synuclein aggregation and neuroinflammation, as seen in PD. The authors further concurred that the gut-brain relationship in PD may be related to SCFAs, curli-producing E. coli , functional amyloids, and alpha-synuclein.

Fabrication of Curli Fiber-PEDOT:PSS Biomaterials with Tunable Self-Healing, Mechanical, and Electrical Properties.

Poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS) is a highly conductive, easily processable, self-healing polymer. It has been shown to be useful in bioelectronic applications, for instance, as a biointerfacing layer for studying brain activity, in biosensitive transistors, and in wearable biosensors. A green and biofriendly method for improving the mechanical properties, biocompatibility, and stability of PEDOT:PSS involves mixing the polymer with a biopolymer. Via structural changes and interactions with PEDOT:PSS, biopolymers have the potential to improve the self-healing ability, flexibility, and electrical conductivity of the composite. In this work, we fabricated novel protein-polymer multifunctional composites by mixing PEDOT:PSS with genetically programmable amyloid curli fibers produced byEscherichia coli bacteria. Curli fibers are among the stiffest protein polymers and, once isolated from bacterial biofilms, can form plastic-like thin films that heal with the addition of water. Curli-PEDOT:PSS composites containing 60% curli fibers exhibited a conductivity 4.5-fold higher than that of pristine PEDOT:PSS. The curli fibers imbued the biocomposites with an immediate water-induced self-healing ability. Further, the addition of curli fibers lowered the Young's and shear moduli of the composites, improving their compatibility for tissue-interfacing applications. Lastly, we showed that genetically engineered fluorescent curli fibers retained their ability to fluoresce within curli-PEDOT:PSS composites. Curli fibers thus allow to modulate a range of properties in conductive PEDOT:PSS composites, broadening the applications of this polymer in biointerfaces and bioelectronics.

Anti-Biofilm and Associated Anti-Virulence Activities of Selected Phytochemical Compounds against Klebsiella pneumoniae.

The ability of Klebsiella pneumoniae to form biofilm renders the pathogen recalcitrant to various antibiotics. The difficulty in managing K. pneumoniae related chronic infections is due to its biofilm-forming ability and associated virulence factors, necessitating the development of efficient strategies to control virulence factors. This study aimed at evaluating the inhibitory potential of selected phytochemical compounds on biofilm-associated virulence factors in K. pneumoniae, as well as authenticating their antibiofilm activity. Five phytochemical compounds (alpha-terpinene, camphene, fisetin, glycitein and phytol) were evaluated for their antibacterial and anti-biofilm-associated virulence factors such as exopolysaccharides, curli fibers, and hypermucoviscosity against carbapenem-resistant and extended-spectrum beta-lactamase-positive K. pneumoniae strains. The antibiofilm potential of these compounds was evaluated at initial cell attachment, microcolony formation and mature biofilm formation, then validated by in situ visualization using scanning electron microscopy (SEM). Exopolysaccharide surface topography was characterized using atomic force microscopy (AFM). The antibacterial activity of the compounds confirmed fisetin as the best anti-carbapenem-resistant K. pneumoniae, demonstrating a minimum inhibitory concentration (MIC) value of 0.0625 mg/mL. Phytol, glycitein and α-terpinene showed MIC values of 0.125 mg/mL for both strains. The assessment of the compounds for anti-virulence activity (exopolysaccharide reduction) revealed an up to 65.91% reduction in phytol and camphene. Atomic force microscopy detected marked differences between the topographies of untreated and treated (camphene and phytol) exopolysaccharides. Curli expression was inhibited at both 0.5 and 1.0 mg/mL by phytol, glycitein, fisetin and quercetin. The hypermucoviscosity was reduced by phytol, glycitein, and fisetin to the shortest mucoid string (1 mm) at 1 mg/mL. Phytol showed the highest antiadhesion activity against carbapenem-resistant and extended-spectrum beta-lactamase-positive K. pneumoniae (54.71% and 50.05%), respectively. Scanning electron microscopy correlated the in vitro findings, with phytol significantly altering the biofilm architecture. Phytol has antibiofilm and antivirulence potential against the highly virulent K. pneumoniae strains, revealing it as a potential lead compound for the management of K. pneumoniae-associated infections.

Extracellular Secretion and Simple Purification of Bacterial Collagen from Escherichia coli

Because of structural similarities with type-I animal collagen, recombinant bacterial collagen-like proteins have been progressively used as a source of collagen for biomaterial applications. However, the intracellular expression combined with current costly and time-consuming chromatography methods for purification makes the large-scale production of recombinant bacterial collagen challenging. Here, we report the use of an adapted secretion pathway, used natively byEscherichia colito secrete curli fibers, for extracellular secretion of the bacterial collagen. We confirmed that a considerable fraction of expressed collagen (∼70%) is being secreted freely into the extracellular medium, with an initial purity of ∼50% in the crude culture supernatant. To simplify the purification of extracellular collagen, we avoided cell lysis and used cross-flow filtration or acid precipitation to concentrate the voluminous supernatant and separate the collagen from impurities. We confirmed that the secreted collagen forms triple helical structures, using Sirius Red staining and circular dichroism. We also detected collagen biomarkers via Raman spectroscopy, further supporting that the recombinant collagen forms a stable triple helical conformation. We further studied the effect of the isolation methods on the morphology and secondary structure, concluding that the final collagen structure is process-dependent. Overall, we show that the curli secretion system can be adapted for extracellular secretion of the bacterial collagen, eliminating the need for cell lysis, which simplifies the collagen isolation process and enables a simple cost-effective method with potential for scale-up.